Project Summary Antibiotic-resistant microbes are once again a major threat to human health worldwide. Drugs once crucial in fighting the spread of deadly pathogens have become ineffective. The development of novel antimicrobials to solve this crisis will require detailed knowledge of the underlying mechanisms of antibiotic resistance, including the structural basis of resistance caused by mutations in antibiotic targets. The most important antibiotic target is the ribosome, the universal site of protein synthesis, and resistanc mutations have been identified in numerous species. Through a strong collaborative effort, this proposal capitalizes on the current state of genetics and X-ray crystallography of the ribosome from Thermus thermophilus to address fundamental aspects of antibiotic resistance. The aims of this proposal are: (1) determine the structural basis for phenotypic interaction among multiple antibiotic-resistance mutations that can potentially arise after sequential exposure to multiple antibiotic challenges; (2) monitor the evolution of secondary mutations that compensate for the fitness cost of deleterious antibiotic-resistance mutations. These aims are designed to give fundamental insights into mechanisms of antibiotic resistance, and findings will be applicable to the rational design of drugs directed against antibiotic-resistant pathogens. Achieving these aims will involve an innovative fusion of genetics and X-ray crystallography to determine the structures of mutant ribosomes. The bacterium T. thermophilus is especially suited as a model system due to its amenability to genetic manipulation and the suitability of its ribosomes for crystallization. Our proposal rests on a strong foundation of: (1) extensive experience in both genetics and X-ray crystallography, including a proven track record of the skills needed to achieve the proposed aims; (2) extensive preliminary results, including the structure determination of a number of antibiotic-resistant ribosomes; (3) an extensive working knowledge of the protein synthesis field and antibiotic-resistance, as indicated by a long track record of publications; (4) an excellent collaborative relationship between the two PIs. The accomplishment of the goals of this proposal will provide fundamental insights into the mechanism of antibiotic resistance, which will have predictive power regarding resistance mutations not yet identified. They will also create a valuable framework for the rational design of novel antimicrobials.